Journal: The Journal of Neuroscience
Article Title: Bovine CNS Myelin Contains Neurite Growth-Inhibitory Activity Associated with Chondroitin Sulfate Proteoglycans
Figure Lengend Snippet: Myelin fractions enriched in proteoglycans do not support cell attachment and neurite outgrowth. Partially purified cerebellar granule neurons were seeded onto nitrocellulose-coated culture dishes absorbed with various myelin fractions as described in Material and Methods and were allowed to grow for 24 hr. The substrates are as follows: A, laminin (10 ÃÂ¼g/ml); B, spinal cord myelin Chaps extract (10 ÃÂ¼g/ml); C, spinal cord myelin Chaps extract (10 ÃÂ¼g/ml) preincubated with the mAB IN-1 (1:1); D, proteoglycan-enriched myelin fraction (5 ÃÂ¼g/ml); E, proteoglycan-enriched myelin fraction (5 ÃÂ¼g/ml) preincubated with the mAB IN-1 (vol 1:1); F , proteoglycan-enriched myelin fraction after chondroitinase ABC treatment. Whereas cell attachment and neurite growth was extensive on laminin, there is reduced adhesion and outgrowth of cells on spinal-cord myelin extract or on a proteoglycan-enriched myelin fraction. Note that the mAB IN-1 could partially neutralize the effect of the spinal cord myelin extract but not of the proteoglycan-enriched myelin fraction. Treatment with chondroitinase ABC partially abolishes the latter activity. G , Quantification of neurite outgrowth of cerebellar granule cells on various substrates. The results are the mean ÃÂ± SEM of five independent experiments. LN , Laminin-1; myelin , total myelin extract; PG , proteoglycan-enriched fraction. Scale bar, 50 ÃÂ¼m.
Article Snippet: Proteoglycan-enriched fractions were digested with protease-free chondroitinase ABC (Boehringer Mannheim, Indianapolis, IN) in 40 m m Tris, pH 8.0, 40 m m sodium acetate, and 0.1 mg/ml BSA.
Techniques: Cell Attachment Assay, Purification, Activity Assay